Abstract

Many components and pathways transducing multifaceted and deleterious effects of stress stimuli remain ill-defined. The Ran-binding protein 2 (RanBP2) interactome modulates the expression of a range of clinical and cell-context-dependent manifestations upon a variety of stressors. We examined the role of Ranbp2 haploinsufficiency on cellular and metabolic manifestations linked to tyrosine-hydroxylase (TH(+)) dopaminergic neurons and glial cells of the brain and retina upon acute challenge to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonian neurotoxin, which models facets of Parkinson disease. MPTP led to stronger akinetic parkinsonism and slower recovery in Ranbp2 (+/-) than wild-type mice without viability changes of brain TH(+)-neurons of either genotype, with the exception of transient nuclear atypia via changes in chromatin condensation of Ranbp2 (+/-) TH(+)-neurons. Conversely, the number of wild-type retinal TH(+)-amacrine neurons compared to Ranbp2 (+/-) underwent milder declines without apoptosis followed by stronger recoveries without neurogenesis. These phenotypes were accompanied by a stronger rise of EdU(+)-proliferative cells and non-proliferative gliosis of GFAP(+)-Müller cells in wild-type than Ranbp2 (+/-) that outlasted the MPTP-insult. Finally, MPTP-treated wild-type and Ranbp2 (+/-) mice present distinct metabolic footprints in the brain or selective regions thereof, such as striatum, that are supportive of RanBP2-mediated regulation of interdependent metabolic pathways of lysine, cholesterol, free-fatty acids, or their β-oxidation. These studies demonstrate contrasting gene-environment phenodeviances and roles of Ranbp2 between dopaminergic and glial cells of the brain and retina upon oxidative stress-elicited signaling and factors triggering a continuum of metabolic and cellular manifestations and proxies linked to oxidative stress, and chorioretinal and neurological disorders such as Parkinson.

MPTP treatment and experimental time line. The baseline motor activities of wild-type and Ranbp2+/− mice were assessed the day prior to the MPTP treatment (Day −1). Wild-type and Ranbp2+/− mice on an inbred 129P2/OlaHsd background underwent four bolus of MPTP i.p. injections spaced 2 h apart. The motor activities of the mice were assessed daily; brain and retinal tissues were collected for analyses at days 6 and 14 after MPTP treatment. Metabolic profiling was performed with brains of wild-type and Ranbp2+/− mice at day 6 after MPTP treatment

Motor activities of wild-type and Ranbp2+/− mice before and after MPTP treatment. a Motor activities between wild-type and Ranbp2+/− mice during the first 7 days after MPTP treatment. Wild-type and Ranbp2+/− mice exhibit no significant differences in basal motor activities a day prior to the initiation of MPTP treatment (day−1). The decline in motility reached its peak at the second day post-treatment and it plateaued for about 2 days before mice of both genotypes began to regain steadily their motilities. The greatest difference in motility between genotypes was observed at day 6 after MPTP treatment. b The recovery of the motility of wild-type mice is significantly faster than Ranbp2+/− mice. Data shown represent the mean ± SD; p < 0.002, n = 5 (a); p = 0.04, n = 6 (b). Black and white bars represent wild-type and Ranbp2+/− mice, respectively. n.s. not significant (p > 0.05); +/+ wild-type, +/−Ranbp2+/−

MPTP elicits a stronger decrease and a slower recovery of the number of retinal TH+-neurons in Ranbp2+/− than wild-type mice. a Comparison of the number of TH+-neurons in the retina between Ranbp2+/− and wild-type mice before and after MPTP treatment (6 and 14 days post-treatment). Note the stronger recovery of retinal TH+-neurons in wild-type mice 14 days after MPTP treatment surpasses the number of TH+-neurons observed in non-treated wild-type or Ranbp2+/− mice. Data shown represent the mean ± SD; n = 6, MPTP-treated mice; n = 3, non-treated mice. b Confocal images of retinal TH+-neurons (red) in non-treated (NT) and 14-day post-MPTP treated wild-type and Ranbp2+/− mice. Note the increase of the thickness of the TH+-synaptic plexus and the presence of weakly stained TH+-cell bodies surrounding a strongly stained TH+-amacrine cell body (inset picture) in wild-type mice 14 days after MPTP challenge. These weakly stained TH+-cell bodies were never observed in retinas of Ranbp2+/− mice. c The increase of the thickness of the TH+-synaptic plexus was accompanied also by a stronger increase of VMAT2 staining (green) in wild-type than Ranbp2+/− mice. +/+ wild-type, +/− Ranbp2+/−; scale bars 25 μm

Ranbp2 haploinsufficiency suppresses MPTP-elicited gliosis in the retina. a Non-treated mice exhibit extremely weak GFAP staining of Müller cells across the retina. In contrast, there is a prominent centrifugal increase of the number of GFAP+-Müller cells in retinas of MPTP-treated wild-type mice that are strongly decreased in Ranbp2+/− mice 14 days after MPTP treatment. Strong GFAP immunoreactivity is also observed in the optic nerve head (ON). Inset pictures denoted by the arrows are high magnifications of boxed peripheral or central regions of the retina. b Quantitative analyses of immunofluorescence intensity of GFAP+-Müller cells in central and peripheral regions of retinas of wild-type and Ranbp2+/− mice 14 days after MPTP treatment (as shown in a). Data shown represent the mean ± SD; n = 3. c The significant differences in the density of GFAP+-Müller cells observed between wild-type mice and Ranbp2+/− mice in a are not accompanied by differences in staining of the canonical glial (Müller) marker, glutamine synthetase, which, in contrast to GFAP, stains uniformly the glial cells across the retina. Inset pictures denoted by the arrows are high magnifications of peripheral or central regions of the retina. +/+ wild-type, +/−Ranbp2+/−, A.U. arbitrary units, ON optic nerve head; scale bars 200 μm

Retinal cellular proliferation elicited by MPTP is suppressed by insufficiency of RanBP2 and it lacks canonical dopaminergic, inflammatory, and vascular cellular markers. a The decrease of the number of TH+-amacrine neurons is accompanied by a strong decrease of EdU+-proliferating cells in Ranbp2+/− compared to wild-type mice 6 days after MPTP treatment. Data shown was collected from images of flat-mount retinas and represent the mean ± SD; n = 4, wild-type; n = 5, Ranbp2+/−. b 3-D reconstruction of confocal x-y-z image stacks across a 50-μm depth of the inner nuclear and plexiform layers of the retina and depicting the non-overlapping spatial arrangement of cell bodies of TH+-amacrine neurons (red) and EdU+-cells (green) 6 days after MPTP treatment in wild-type and Ranbp2+/− mice. Ranbp2+/− mice present far fewer EdU+-cells than do wild-type mice. The images below the 3-D images are 2-D cross sections of collapsed 3-D images. Scale bars: x 635.5 μm, y 635.5 μm, z 50 μm with cell bodies of TH+-amacrine neurons occupying the mid-plane of the z-stack. c Only few GFAP+-cells (red) in the retina were co-labeled by EdU (green) in wild-type mice; GFAP+-cells are shown with (arrow) and without (arrowhead) EdU labeling. d No CD45+-cells (red, arrowhead) in the retina were observed to be labeled by EdU (green, arrow) in mice of either genotype, although in the choroid plexus few CD45+-cells were co-labeled by EdU (arrow) (e). f No Sox2+-cells (red, arrowhead) in the retina were observed to be labeled by EdU (green, arrow) in mice of either genotype. Images c–f are representative retinal sections of mice provided with a daily EdU bolus for 13 days after MPTP treatment. +/+ wild-type, +/− Ranbp2+/−

Model of metabolic deregulation in the brain by RanBP2 insufficiency upon MPTP insult. Alterations of brain metabolites by RanBP2 insufficiency upon acute MPTP exposure were subdivided into five major groups (a–e). Metabolites of group a participate in depicted multistep and interdependent metabolic pathways regulating bioenergetics in the cytosol or mitochondria related to lipid and glucose metabolism and cofactors thereof. These metabolites can be further divided into three subsets: lipid metabolites or precursors and cofactors thereof, acylcarnitine conjugates and metabolites of lysine catabolism, bioenergetic metabolites/substrates, and cofactors thereof (e.g., glycerophosphorylcholine, glucose, lactate, NAD+) that likely promote reduced anaplerosis of the Krebs cycle. Metabolites of group b reflect changes in histidine catabolic pathways, whereas those of group c represent variations in the methionine and adenine salvage pathways. Metabolites of groups d and e represent changes in conjugation reactions with glycine, reflecting variations of redox pathways (group d) and detoxification reactions (group e). See text for further details. Metabolites in red and green are, respectively, up-regulated and down-regulated; single and multiple arrows represent single and multi-step pathways. MAO-B monoamine oxidase, type BORC1 ornithine carrier, CPTII carnitine palmitoyltransferase II, ODC oxodicarboxylate carrier, OGC oxoglutarate carrier